Such assays are normally prepared and measured in sample plates or formats, including 96-well microtitre plates, petri dishes, gel matrices, membranes, glass slides and capillaries. The trend is towards higher throughput detection of samples and the use of smaller volumes in each of the samples, resulting in so-called miniaturised sample formats.
This requires the corresponding development of detectors capable of handling such miniaturised formats. This is particularly so in the case of high throughput screening (HTS) of biological assays as applied to drug discovery and the screening of drug candidates.
Such miniaturisation is achieved by arranging samples for assay and detection in well plates in which typically there can be 96, 384, 864, 1536 or 3456 wells per plate, and sample volumes can vary from 200 microlitres to as little as 1 microlitre.
Alternative formats for the miniaturisation of assays include capillaries, microchannels or microfluidic structures, including microwells, which can be moulded or etched in substrates such as glass (eg silica or quartz) or plastic. In these alternative formats, the sample volumes can be of the order of nanolitres and picolitres.
In order to achieve high throughput screening it is necessary to interrogate large numbers of such samples simultaneously. In the case of fluorescence based assays, detection or interrogation consists of illuminating each sample with excitation light, and subsequently detecting the emitted fluorescence from each sample separately. Examples of fluorescent based processes include prompt fluorescence and time-resolved fluorescence where there is a time delay between photoactivation of the sample and emission, in the range, for example of ps to ms or more. A further process is fluorescence or luminescence energy transfer. In this process a molecule is activated, for example by excitation light, and transfers energy via eg resonance energy transfer or chemical transfer to a second molecule, which in turn emits light. This process can involve different or multiple secondary molecules, which can emit radiation over a range of wavelengths. Further examples of luminescent processes include phosphorescence, and chemi- and bio-luminescence. Wavelength ranges for all these processes include UV, visible, red and infra-red (approx 250–1200 nm).
In a typical arrangement, a scanning head with 96-channels simultaneously interrogates the 96-sites arranged in the 8×12 pattern of a 96-well microtitre plate. By stepping a larger well density plate relative to the 96-channel scanning head, the remaining sites can be read eg 2×2 steps will cover 384-samples, 6×6 steps will cover 3456 samples, and so on, so as to address a high density sample presentation format.
Fluid samples, eg liquids or gels, can be placed in small sample sites, such as micro-capillaries or microchannels, which can be typically 100×100 um in section, and typically 1–100 mm long. The samples can be moved by pumps, or electrophoretically or electro-osmotically. Samples can be solid or a matrix, such as beads, agarose or microparticles, suspended or otherwise contained in a fluid medium, including a gel type format. Other samples can comprises suspensions or monolayers of cells.
Applications include cell biology, hybridisation techniques and immunoassays including binding assays. In such assays, materials can be labelled with a fluorescence marker for the purpose of identification. In a binding assay, a bound molecule can be separated from an unbound molecule, as between a solid and liquid medium. Further applications include electrophoresis or electro-osmosis fluids such as liquids in gels and media including agarose. Such applications can be run in miniaturised formats including micro-capillaries and micro-fluidic structures, where molecules of moities may be separated spatially by properties including molecular weight or charge and may also be labelled with fluorescent or luminescent tags or dyes for the purpose of detection and identification. The techniques described herein can be applied to the detection of biological compounds including proteins and nucleic acids, and in cell biology, processes such as cell signalling or cell binding can also be detected.
Separated molecules can be contained in a fixed matrix, such as a gel or agarose beads, or can be separated in a fluid such that the separated molecules or moities will flow at different rates through a medium and can be detected at a fixed point along the flow path as a series of emission peaks based on their time separation profile. It is important that emission detection methods posses high accuracy and sensitivity for the determination of such peaks, and the rapid and/or continuous and/or simultaneous measurement of samples.
A number of such assay techniques involve time changes in light emission. To measure such changes requires an ability to perform rapid, accurate, sensitive and repetitive readings, that is to perform kinetic measurements. In order to achieve high throughput it is necessary for a system to measure multiple samples simultaneously, requiring high resolution, high sensitivity and high signal detection efficiency.
Mostly the samples/assays are arranged to emit light in the middle range, although the quantities of light per sample can be very low.
A further issue with fluorescence-based assays is the problem of quench which causes a reduction in light emission. This can occur in samples and assays particularly those involving cells, due to chemical effects which interfere with the light signal, or due to coloured substances or particles in the sample or medium, which reduce the light signal. In certain applications, such as inhibition assays, a change or reduction in light signal is the feature of the assay requiring measurement.
FIG. 1(a) of the accompanying drawings shows an array of 8×12 micro capillaries in a substrate, in which either a liquid, or molecules or moities in a fluid, move in the X-direction. This is an example of an array of miniaturised samples such as will be referred to in this application.
FIGS. 1(b) and 1(c) of the accompanying drawings show further examples of arrays that involve high density or miniaturised sample formats.
A confocal microscope is a means for achieving excellent imaging capability whilst possessing a unique depth-discriminating property. (A typical Zeiss system is shown in FIG. 15.5 in “Quantitative fluorescence microscopy”, F. W. D. Rost, CUP 1991). A parallel light beam from a laser or other suitable light source can be used for fluorescence excitation. The beam is focused by a microscope objective, down to a spot of typically micron or submicron size, within the sample. The fluorescent light emanating from the spot is focused by the same objective, via a beam splitter, onto a pinhole aperture (a spatial filter), and thence onto the detector. The detector does not accept all the light from out-of-focus planes and so these are imaged less strongly by the spatial filter. Thus detected background fluorescence from the substrate or material surrounding the sample, which is also unavoidingly illuminated, is sharply reduced relative to the wanted signal. Moving the microscope relative to the plurality of sample spots or vice versa, allows the spots to be inspected in sequence or scanned.
A confocal microscope therefore has the essential attributes for interrogating the tiny samples described above, such as micro capillaries.
Firstly, there is excellent spatial resolution to excite a small region of a sample and avoid exciting laterally surrounding material.
Secondly, it has good depth discrimination, which minimises the effects of illumination of the substrate, above and below the sample. Hence the effect of background fluorescence at the detector can be reduced/minimised.
Single channel scanners for the purpose have been reported in the literature. For example, we refer to “DNA sequencing using Capillary array Electrophoresis” by X C Huang, M A Quesada and R A Mathies, Anal. Chem. 1992, 64, 2149–2154. In their apparatus a 32×. NA 0.4, Carl Zeiss microscope objective brings the parallel beam from a 1 mW argon ion laser to a 10 micron diameter spot, within a 100 micron id sample capillary. The fluorescence is collected by the same objective, passed via a beam splitter and, after wavelength selection via spectral filters, is focused onto a 400 micron diameter pinhole. The spatially filtered light is then detected by a photomultiplier. In this apparatus a set of capillaries mounted side by side is interrogated successively by the confocal microscope, by means of relative XY movement between the samples and the microscope. However a serious disadvantage of this approach is that no sample is read continuously but is read at best, on a periodic basis. Typically any one sample can be read about once per second.
Thus a single channel confocal scanner has been used to measure a number of sample sites but only sequentially and not simultaneously, and such a system is not appropriate for high throughput scanning where simultaneous detection and assessment of a large number of sites is required, such as in a 96 well microtitre plate or higher density format array or array of microcapillaries.
However, there is known from German Specification No DE 19748211A a system in which a plurality (N) of single channel confocal optical systems and photoelectric detectors or detecting areas are arranged in parallel to form a plurality of reading heads arranged side-by-side so as simultaneously to read a corresponding plurality of adjacent sites. In this known system, the optics include a mask and three focussing lenses which effectively handle the beams emanating from the individual reading heads independently, so that the optical axes of the individual beams are separated.